Fig 2: Analysis of the invasive front of human primary melanomas.a–i Representative images (left) and quantification (right) of a melanoma cell shape score, b H-score of p-MLC2 staining, c ki-67 positive cells, H-score of d DAAM1, e WNT11, f WNT5B, g ALDH1A1, h CD44 and i NANOG staining in matched TB and IF from primary melanomas. j Principal component analysis (PCA) based on the expression of amoeboid (cell shape and p-MLC2), proliferative (ki-67), non-canonical Wnt pathway (WNT11, WNT5B and DAAM1) and cancer stem cell-related (ALDH1A1, CD44 and NANOG) markers assessed by immunohistochemistry in matched TB and IF from primary melanomas. Percentage of variation explained by each component is given in the axis labels. k, l Kaplan–Meier survival curves of k overall survival and l disease-free survival according to ALDH1A1 protein expression in the IF from our cohort of primary melanomas. ALDH1A1 expression was categorized as low or high using the median expression. a–l 53 primary melanomas. a, e–i Scale bar, 100 μm; inset, 50 μm. b–d Scale bar, 300 μm. a–i Box limits show 25th and 75th percentiles, the horizontal line shows the median, and whiskers show the minimum and maximum range of values. a, b, d–f Two-tailed paired t-test. c, g–i Two-tailed Wilcoxon test. j Scatter plot showing principal components 1 (PC1) and 2 (PC2). k, l Log-rank test. Human schematic in this figure was created using Servier Medical Art templates licensed under a Creative Commons Attribution 3.0 Unported License (https://smart.servier.com).

Fig 3: Analysis of the invasive front of human metastatic melanomas.a–i Representative images (left) and quantification (right) of a melanoma cell shape score, b H-score of p-MLC2 staining, c ki-67 positive cells, H-score of d DAAM1, e WNT11, f WNT5B, g ALDH1A1, h CD44 and i NANOG staining in matched TB and IF from melanoma metastases. j Model summarizing the findings of this study. The IF of human primary melanomas is enriched in cells with rounded-amoeboid morphology, high levels of Myosin II, proliferative, non-canonical Wnt and cancer stem cell-related markers. This phenotype is recapitulated and further enriched in melanoma metastasis. WNT11/5B activate FZD7 and DAAM1 to control Rho activity and then ROCK1/2-Myosin II levels. All these signalling components have an impact on amoeboid features and tumour initiation in melanoma both in vitro and in vivo. Specifically, DAAM1 plays an essential role in tumour and metastasis initiation and subsequent metastatic outgrowth by sustaining the amoeboid phenotype. a–i 45 metastatic melanomas. a, e–i Scale bar, 100 μm; inset, 50 μm. b–d Scale bar, 300 μm. a–i Box limits show 25th and 75th percentiles, the horizontal line shows the median, and whiskers show minimum and maximum range of values. a, b, d–f Two-tailed paired t-test. c, g–i Two-tailed Wilcoxon test. Human schematic in this figure was created using Servier Medical Art templates licensed under a Creative Commons Attribution 3.0 Unported License (https://smart.servier.com).

Fig 4: Non-canonical Wnt ligands support melanosphere formation and amoeboid behaviour.a mRNA expression of stem cell-related markers by qRT-PCR in A375M2 cells treated with ROCKi (GSK269962A) for 24 h compared to control A375M2 cells (n = 4 for ALCAM, ALDH1A3, ALDH1A1; n = 3 for CD44, OCT4, JARID1B, SOX2). b Schematic of treatment (top), representative phase-contrast images (bottom left) and quantification of sphere formation index (bottom right) and c cell viability of A375M2 cells and A375M2 cells treated with one dose of ROCKi (H1152 or GSK269962A) or blebbistatin (n = 3). Scale bar, 250 μm. d–f After ROCK1/2, MYL9 or MYL12B knockdown in A375M2 and WM1361 cells, quantification of d sphere formation index (n = 4 for A375M2, n = 3 for WM1361) and e, f cell viability after e 7 days (n = 3 for A375M2, n = 4 for WM1361) or f 3 days (n = 3) of cell seeding. g–j After WNT11 or WNT5B knockdown in A375M2 and WM1361 cells, g, h representative phase-contrast images (left) and quantification of sphere formation index (right) (n = 3) and i, j cell viability (n = 4 for A375M2, n = 3 for WM1361). Scale bar, 250 μm. k Representative phase-contrast images (left) and quantification of cell morphology (right) of A375P cells after 24 h of WNT11 or WNT5B stimulation (>225 cells pooled from n = 3). Scale bar, 100 μm. a–j Graphs show mean ± s.e.m. k Box limits show 25th and 75th percentiles, the horizontal line shows the median, and whiskers show minimum and maximum range of values. a–k n means number of independent biological experiments. a–c, h, j Two-tailed t-test. d–g, i One-way ANOVA with Dunnett post-hoc test. k Kruskal–Wallis with Dunn’s multiple comparison test. For all graphs, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. The exact significant p values for *p, **p and ***p are provided in Supplementary Table 1.

Fig 5: Pyrvinium targets multiple vulnerabilities of MCC.(A) Protein levels of WNT5A/B under pyrvinium treatment for 24 hrs in WaGa and MKL-2 cells. (B) Relative mRNA levels of AXIN2, ATOH1 and SOX2 genes in WaGa cells after treatment with human recombinant WNT5B protein for 6 hrs, measured by RT-qPCR. Statistical significance was determined by unpaired two sample T-test. (C) Protein levels of total β-catenin, WNT5B, ATOH1, SOX2 and GFP after treatment with human recombinant WNT5B for 6 hrs in WaGa TopGFP stable cells. (D) Protein levels of p53, intrinsic apoptosis pathway indicator cleaved-PARP and pro-apoptotic protein PUMA were measured by WB in p53 wild type cell lines (WaGa, MKL-1) and p53 mutant/null cell lines (MS-1, MKL-2) 24 hours post 0.5 μM of pyrvinium treatment and (E) 1 μM of Nutlin-3a treatment respectively. (F) Representative data shown as a line chart to demonstrate the basal respiration level and maximal respiration capacity (after FCCP injection) at each measurement time point (means ± SEM; n=6). (G) Seahorse OCR analysis was performed to measure uncoupled OCR in WaGa cells treated with different doses of pyrvinium for 24 h, compared with cells treated with DMSO. Statistical significance was determined Kruskal Wallis H Test followed by Dunnett’s post hoc test. (H) Quantification of WB results of all batches of OXPHOS WB result (n = 4). The mean relative protein expression of GAPDH in vehicle control samples was taken as 100%, with all other values expressed relative to this 100% (means ± SEM). Statistical significance was determined by ordinary ANOVA test followed by Dunnett’s multiple comparison test. (I) The level of proteins that reflect ER stress was detected by WB in MCC cell lines (WaGa, MKL-1, MS-1, and MKL-2) under treatment by pyrvinium for 6 hours and 24 hours. (J) The level of proteins that reflect UPR response was detected by WB in WaGa cells after treatment with pyrvinium or other ER stress inducers for 24 hours. (****, p < 0.0001; ***, p < 0.001; **, p < 0.01; *, p < 0.05)